Local Injection of Deferoxamine Improves Neovascularization in Ischemic Diabetic Random Flap by Increasing HIF-1? and VEGF Expression.

OPEN Q ACCESS Freely available online

PLOS | ONE

Local Injection of Deferoxamine Improves (Gt\
Neovascularization in Ischemic Diabetic Random Flap by c^m**
Increasing HIF-1a and VEGF Expression

Chen Wang 19 , Yuanyuan Cai 2< *, Yun Zhang 1 , Zhuyou Xiong 3 , Guangzao Li 3 *, Lei Cui 1 *

1 Department of Plastic and Reconstructive Surgery, Shanghai 9 th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China,

2 Department of Plastic and Aesthetic Surgery, Changzhou NO.2 People's Hospital, Changzhou, P. R. China, 3 Department of Plastic Surgery, 1 st Affiliated Hospital of
Bengbu Medical College, Bengbu, Anhui, P. R. China

Abstract

Background: Although the systemic administration of deferoxamine (DFO) is protective in experimental models of normal
ischemic flap and diabetic wound, its effect on diabetic flap ischemia using a local injection remains unknown.

Objective: To explore the feasibility of local injection of DFO to improve the survival of ischemic random skin flaps in
streptozotocin (STZ)-induced diabetic mice.

Methods: Ischemic random skin flaps were made in 125 mice. Animals were divided into the DFO-treated (n = 20), PBS-
treated (n = 16) and untreated (n = 16) groups. Surviving area, vessel density, and expression of vascular endothelial growth
factor (VEGF) and hypoxia-inducible factor-lot (HIF-1 oc) were evaluated on the seventh day after local injection.

Resu/ts:Jhe viability of DFO-treated flap was significantly enhanced, with increased regional blood perfusion and capillary
density compared with those in the two control groups. Fluorescence-activated cell sorting (FACS) analysis demonstrated a
marked increase in systemic Flk-1 + /CD1 1b~ endothelial progenitor cells (EPCs) in DFO-treated mice. Furthermore, the
expression of VEGF and HIF-1 oc was increased not only in diabetic flap tissue, but also in dermal fibroblasts cultured under
hyperglycemic and hypoxic conditions.

Conclusions: Local injection of DFO could exert preventive effects against skin flap necrosis in STZ-induced diabetic mice by
elevating the expression of HIF-1 ex and VEGF, increased EPC mobilization, which all contributed to promote ischemic
diabetic flap survival.

Citation: Wang C, Cai Y, Zhang Y, Xiong Z, Li G, et al. (2014) Local Injection of Deferoxamine Improves Neovascularization in Ischemic Diabetic Random Flap by
Increasing HIF-1a and VEGF Expression. PLoS ONE 9(6): e100818. doi:10.1371/journal.pone.0100818

Editor: Michael E. Boulton, Indiana University College of Medicine, United States of America

Received February 13, 2014; Accepted May 29, 2014; Published June 25, 2014

Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Natural Science Foundation of China (Grant No: 81 201 204) and the Natural Science Foundation of Shanghai (Grant No:
1 1ZR1 420300). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* Email: liguangzhaoby_jy@163.com (GL); cuileijy@163.com (LC)

<3 These authors contributed equally to this work.

Introduction

In diabetic patients, the management of impaired healing of
cutaneous wounds, such as foot and leg ulcers, represents a
significant public health burden worldwide [1,2]. Random skin
flaps are used for treating these wounds and ulcers. However,
necrosis often occurs in the distal part of random skin flaps in
diabetic individuals [3-5], which has been attributed to impaired
ischemia-driven neovascularization under hyperglycemic condi-
tions. Local administration of angiogenic growth factors, such as
vascular endothelial growth factor (VEGF) [6], fibroblast growth
factor-2 (FGF-2) [7] , and adult mesenchymal stem cells from either
bone marrow or adipose tissue, has been documented to be
effective in improving neovascularization in diabetic random flaps
[8,9] . However, the safety and use of these approaches remain
controversial [10], and no efficient therapy is currently available in
a clinical setting.

Deferoxamine (DFO), a free radical scavenger and iron
chelator, has been shown to improve skin flap survival by up-
regulating VEGF in ischemic flap surgery [11-14]. As a key
transcription factor, hypoxia-inducible factor- 1(X (HIF-1 a) is
necessary for the expression of angiogenic growth factors like
VEGF, and for endothelial progenitor cells (EPCs) recruitment to
ischemic sites in order to form new blood vessels [15]. Recently, it
was found that the systemic use of DFO increased HIF-1 a
stabilization and improved age-related decline in HIF-loc
[3,11,15]. In addition, HIF-1 (X activity was corrected with DFO
administration by preventing iron-catalyzed reactive oxygen
species (ROS) and methylglyoxal formation under hyperglycemia
[3,16,17]. However, it is still unclear if the local administration of
DFO could correct HIF- 1 oc and VEGF expression in a hypergly-
cemic environment, and enhance the viability of diabetic random
skin flaps.

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Effects of DFO on Diabetic Random Flap

In the present study, ischemic random skin flaps were made in
streptozotocin (STZ)-induced diabetic mice, and DFO was
injected locally in the skin flap to investigate whether tissue
necrosis could be rescued after improved angiogenesis. Further-
more, expression of HIF-lcx and VEGF was detected after DFO
treatment in skin flap tissue and in skin fibroblasts cultured under
high glucose and hypoxic conditions. Finally, EPCs mobilization
in the peripheral circulation was evaluated in response to DFO
administration.

Materials and Methods

Development of ischemic random skin flap in diabetic
murine model

One hundred- twenty-five C57/bl6 male mice aged 8—
10 weeks and weighing 20-25 g were purchased from SLAC
National Rodent Laboratory Animal Resources (Shanghai,
China). This study was carried out in strict accordance with
the recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
institutional review committee of the Shanghai Jiao Tong
University School of Medicine approved all animal study
protocols. All mice were anesthetized with intraperitoneal
injections of pentobarbital sodium (20 mg/kg body weight) in
all surgical procedures, and all efforts were made to minimize
suffering. Diabetes was induced by intraperitoneal injections of
streptozocin (50 mg/kg, Sigma-Aldrich, St Louis, MO, USA) in
50 mM sodium citrate buffer (pH 4.5) for 5 consecutive days
[18]. Venous blood was collected from the tail for blood glucose
levels determination 14 days after the first day with STZ
injection using a OneTouch Ultra portable glucose analyzer
(Lifescan Inc., Milpitas, CA, USA). Diabetic mouse model was
considered to be successful when glucose levels were above
300mg/dl and in the presence of weight loss and polydipsia,
polyphagia, and polyuria symptoms.

After diabetes induction, the back's hairs of mice was cut off and
soaked the remaining hair with water and daub with depilatory
cream (Shibi, Shanghai, China) uniformly. Three minutes later,
we erased the cream and repeated the procedure three times, the
hair was removed completely and a cranially-based peninsular skin
flap (1.0x3.0 cm) was elevated in the dorsum of diabetic mice
[19,20], in which the ischemic gradient was proportional to the
distance from the base. The skin was gently stretched to show the
cutaneous vessels (Fig. 1A), and the cutaneous vascular architec-
ture was outlined using a marker pen (Fig. IB). A thin sterile
silicon sheet was inserted to separate the flap from the underlying
bed, and the flap was sutured in place (Fig. 1C). Previous studies
showed that a gradient of oxygen tensions could be generated
within the flap tissue [15].

Local injection of DFO

DFO dissolved in PBS (0.1 ml) was injected subcutaneously in
the distal portion of the flap. Different concentrations of DFO (0,
10, 40, 70, 100, 150 mg/kg) were injected in the flap (Fig. 2A).
The animals were divided into 3 groups: the DFO group, the PBS
group and the untreated group. After flap elevation, DFO
(100 mg/kg, 10 mg/ml, Sigma, St Louis, MO, USA) was
immediately injected in the DFO group, and every day for three
days after surgery (Fig. 3 A). The same amount of PBS was
subcutaneously injected in the PBS group.

Assessment of the surviving areas of flaps and skin blood
perfusion

Seven days after operation, pictures of the flaps were taken
using a digital camera (HP M425, Hewlett-Packard, Palo Alto,
CA, USA). Surviving area of flaps was assessed blindly by two
specialists with respect to gross appearance, color, consistency of
the flaps and presence or absence of bleeding. The surface area of
these defined zones was then measured using the Image-Pro Plus
Software 6.0 (Media Cybernetics, Silver Spring, MD, USA).

Blood perfusion of the skin flap was detected with a laser
Doppler perfusion imaging system (Moor Instruments, Axminster,
UK) 7 days after surgery. The probe was placed on the proximal
necrosis line and 0.5 cm from the distal necrosis edge of the flap
for at least 30 s, and the results were recorded as blood perfusion
units (PU).

Histology and immunofluorescence

Seven days after surgery, seven-micron-thick tissue samples
were harvested from the similar position of the flaps the flap, fixed
in 4% paraformaldehyde, embedded in paraffin, and stained with
hematoxylin and eosin (H&E). For immunohistochemistry, sam-
ples from flap tissues were snap-frozen in liquid nitrogen; seven-
micron-thick frozen sections were fixed in cold acetone and
stained with rat monoclonal anti-CD 31 (1:50, ab7388, Abeam,
Cambridge, MA, USA) primary antibody, followed by addition of
FITC -conjugated secondary antibody (1:1000, A- 11006, Invitro-
gen Inc., Carlsbad, CA, USA). On one hand, neovascularization
was assessed by measuring the number of capillaries in 20 fields in
each mouse on H&E stained slides with high power field (HP) in
each group, on the another hand, vascular density was examined
under fluorescence microscope (Olympus, Tokyo, Japan) and was
assessed by measuring the number of CD31+ cells in frozen
sections. Capillary density was assessed morphometrically by
examining three fields per section of the flap in six successive
sections on both the H&E sections and the immunofluorescence
staining sections after immunofluorescence staining for endothelial
cells with an anti-CD 31 antibody. All measurements were
performed by two blinded reviewers.

Western Blot Analysis

Cells from flaps were processed by extracting proteins with a
lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM
NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% deoxycholate).
Proteins were separated by 8% polyacrylamide gel electropho-
resis containing 0.1% SDS and subsequently transferred to
nitrocellulose membranes. Membranes were blocked with 2%
non-fat dry milk and 3% BSA in Tris-buffered saline. Rabbit
anti-HIF-1 alpha (NB 100- 134; Novus Biologicals, Littleton, CO,
USA), or rabbit anti-VEGF (ab46154; Abeam, Cambridge, MA,
USA) were added. Blots were developed using an IRDye
700DX- and IRDye 800CW-conjugated secondary antibody
(Rockland Immunochemical, Inc., Gilbertsville, PA, USA), and
proteins were visualized using the Odyssey system (LI-COR
Biosciences, Lincoln, NE, USA).

Culture of dermal fibroblasts

Fibroblasts were harvested from STZ-induced diabetic (n = 6,
8—10 weeks old) and non-diabetic mice (n = 6, 8-10 weeks old).
Animals were sacrificed and the trunk skin was removed by sharp
dissection. Special care was taken to remove the underlying
adipose tissue.

Skin biopsies were harvested from diabetic and non-diabetic
mice (n = 6 in each group). After being washed intensively with

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Effects of DFO on Diabetic Random Flap

Figure 1. Vascular pattern of mouse dorsal skin and elevation of the skin flap. (A) The skin was gently stretched to show the cutaneous
vessels. (B) Outlined cutaneous vascular architecture showing that the skin was supplied by 4 major pedicles arising from the deep circumflex iliac
arteries (I) and the lateral thoracic arteries (T). (C) Ischemic flap measuring 1.0x3.0 cm was elevated and a thin silicon sheet was inserted to separate
the flap from the bed. The black arrow indicates the silicone sheet.
doi:1 0.1 371/journal.pone.01 0081 8.g001

0.1 M phosphate buffered saline (PBS, pH 7.4), skin tissue was
minced and digested with 0.1% collagenase type I (Worthington
Biochemical Corp., Lakewood, NJ, USA) at 37°C for 2 hours.
Cells were then centrifuged at 600 g for 10 min and filtered
through a 100-jim nylon mesh to remove undigested tissue. Cells
were resuspended in Dulbecco's modified Eagle's medium
(DMEM, GIBCO, Invitrogen Inc., Carlsbad, CA, USA) supple-
mented with 10% fetal bovine serum (FBS, Hyclone, Thermo
Fisher Scientific, Waltham, MA, USA) and 1% antibiotics/
antimycotics. Experiments were performed using cultured cells at
the third passage. Fibroblasts from diabetic mice were maintained
in high glucose medium (25 mM D-glucose) and subjected to
hypoxia (1% 0 2 , 5% C0 2 ) to mimic the pathological diabetic
parameters in vivo [9]. Dermal fibroblasts from diabetic or non-
diabetic mice were treated with 100 jiM DFO for 48 h, and cell
lysates were used for western blotting [21,22].

Mouse EPC mobilization assay

Peripheral blood (0.4-0.6 ml) was obtained from mice in three
different group (including normal phase (C57/bl6 male mice
without any treatment), DM phase (diabetes was induced by

DFO(mg/kg)

Figure 2. Dose- and time-dependent effects of DFO adminis-
tration on the survival of diabetic skin flap. (A) Dose-dependent
augmentation of surviving area of diabetic flaps upon local injection of
DFO. Results are shown as means ± SEM. *P<0.01 vs. the 0-70 mg/kg
groups. (B) Surviving area at different time points of diabetic flaps with
injection of 100 mg/kg DFO each day. Results are shown as means ±
SEM. *P>0.05. n = 6.
doi:1 0.1 371 /journal.pone.01 0081 8.g002

intraperitoneal injections of strep tozocin for 5 consecutive days)
and DFO+DM phase (after diabetes induction, a skin flap was
elevated in the dorsum of diabetic mice, and DFO (100 mg/kg)
was injected subcutaneously in the distal portion of the flap for
three days) (n = 5). Erythrocytes were lysed with ammonium
chloride and separated by centrifugation. Cells were then washed
with PBS/EDTA and marked using PE-labeled Flk-1 (VEGFR-2;
eBioscience, San Diego, CA, USA) and FITC-labeled CD lib
(eBioscience, San Diego, CA, USA) antibodies. EPCs were
identified as Flk-1+/CD1 lb- [23]. Then, EPC mobilization was
analyzed by flow cytometry (Becton Dickson, San Jose, CA, USA).

Statistical Analysis

Results are expressed as means ± SEM. SPSS 10.0 (SPSS Inc.,
Chicago, IL, USA) was used for statistical analysis. Analysis of
Variance (ANOVA) assuming equal variance (Student-Newman-
Keul test) was performed to identify treatments different from
control group, and p-values <0.05 were considered statistically
significant.

Results

DFO improved flap viability in a dose-dependent manner

The flaps were treated with different doses of DFO and their
surviving area was calculated 7 days after injection. In diabetic
untreated and PBS-treated mice, severe and extended necrosis in
the distal part of the flap was observed at 7 days postoperatively,
accounting for 62.19±9.58% and 61.64±7.63% of the total flap
area, respectively. In DFO-treated diabetic mice, there was a dose-
dependent augmentation of the surviving area, being 65.83%,
83.02%, 90.10% and 89.74% at doses of 10 mg/kg, 70 mg/kg,
100 mg/kg and 150 mg/kg, respectively (P<0.01 compared with
PBS-treated flaps; n = 6; Fig. 2A). In addition, there was no
significant difference in surviving area between the 100 mg/kg
and 150 mg/kg groups (P>0.05). Therefore, the DFO dose was
fixed at 100 mg/kg for the subsequent experiments.

To find a minimal duration for injection, surviving area of skin
flaps was evaluated with daily administration of DFO at 100 mg/
kg for 8 days after flap elevation. The surviving area reached its
peak after 2 days of DFO injection, and the subsequent DFO
injections from days 3 to 8 showed no significant improvement in
surviving area (Fig. 2B). This result indicated that DFO treatment

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Figure 3. Analysis of necrosis and blood flow perfusion of the skin flap. (A) Schematic illustration of DFO injection procedure and evaluation
of skin flaps. (B) Gross view and color laser Doppler detection of diabetic skin flaps from the DFO, PBS and control groups 7 days postoperation. The
color scale illustrates variations in the blood flow, from maximal (red) to minimal perfusion (dark blue). (C) Quantitative analysis of blood flow
perfusion of the flap measured as mean perfusion units ± SEM (*P<0.01). DFO-treated (n = 20), PBS-treated (n = 16) and Blank group (n = 16).
doi:1 0.1 371 /journal. pone.01 0081 8.g003

initiated 1 day before and continued for 3 days after flap elevation
at a dose of 100 mg/kg/day was sufficient to obtain an optimal
protection against necrosis in diabetic random skin flaps.

DFO improved flap viability by increasing blood
perfusion

Using color laser Doppler imaging system analysis, it was found
that blood perfusion to the distal part of the skin flaps remained
lower in the untreated (396.42+14.28 PU) and PBS-treated
groups (41 7.53 ± 14.89 PU) 7 days after surgery, compared with
DFO-treated mice (565.03± 13.15 PU) (P<0.01) (Fig. 3).

DFO improved neovascularization in ischemic skin flaps

As it was reported that DFO promotes survival of ischemic skin
flap through neovascularization, we investigated the distribution of
capillaries in diabetic skin flaps. As shown in Figure 4, samples
from the DFO-treated group displayed an increased distribution of
capillary vessels, which exhibited dilated lumen compared with
vessels in the untreated and PBS-treated groups. Furthermore,
CD31 -positive cells were more abundant in DFO-treated flaps.

DFO enhanced production of VEGF and HIF-1a in
diabetic skin flaps

Many studies have documented a decreased VEGF production
in ischemic diabetic wounds, but it is still unclear if the improved
neovascularization is the result of increased VEGF expression with
administration of DFO in diabetic skin flaps. Thus, expression of
VEGF and HIF- 1 a was examined by western blotting. As shown
in Figure 5, there was no detectable difference in VEGF and HIF-
Icl expression in the intact skin from each group. However,
compared with non-diabetic mice, the expression of VEGF and
HIF- lot was dramatically decreased in untreated and PBS-treated
flaps (all P<0.01). With the administration of DFO, expression of
VEGF and HIF- 1 a was higher, and was similar to that of non-
diabetic normal flaps (Fig. 5).

DFO restored HIF-1a and VEGF expression in diabetic
dermal fibroblasts

Because dermal fibroblasts are the major actors for the healing
of cutaneous wounds, the effect of DFO on HIF-1(X and VEGF
expression in dermal fibroblasts cultured under high glucose and
hypoxic conditions was assessed. Fibroblasts were grown in either
low (5 mM D-glucose) or high glucose (25 raM D-glucose)
medium for 2 weeks, and were then exposed to normoxic or
hypoxic conditions for 3 days. Under hypoxic conditions, DFO
treatment resulted in a 1.7 -fold and 1.8-fold increase in HIF- lex
and VEGF expression, respectively, in cells cultured in high
glucose medium, approximating the levels in cells cultured in low
glucose medium (Fig. 6). Under normoxia (21% 0 2 ), HIF-loc and
VEGF levels in cells cultured in high glucose medium showed an
increase that was similar to the cells cultured in low glucose
medium with the DFO administration (Fig. 6 A, C).

DFO promoted increased EPC mobilization

To examine whether diabetic EPCs retained the ability to
respond to DFO, EPGs from the peripheral blood were analyzed
by FACS. Diabetic mice exhibited a marked decrease in systemic
Flk-1 /CD lib progenitor cells compared with non-diabetic
mice, indicating a specific impairment of vasculogenesis in diabetic
mice (6.34±0.32% vs. 0.42±0.06%, P<0.01; Fig. 7B). With DFO
treatment, FACS analysis demonstrated a marked increase in
systemic Flk-l + /CDllb EPCs in diabetic mice compared with
controls (1.85±0.12% vs. 0.42±0.06% EPCs, P<0.05) (Fig. 7A
B). Thus, increased EPC mobilization was induced by DFO
administration and may contribute in part to the increased
neovascularization of the ischemic flap in diabetic mice.

Discussion

In the present study, we showed that the local injection of DFO
improved the viability of random skin flaps in STZ-induced
diabetic mice, which was accompanied by increased capillary
density in necrotic area. Expression of VEGF and HIF- lex was also

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Figure 4. Determination of capillary density in diabetic flap 7 days after surgery. (A) Gross view, H&E staining and CD31 immunostaining
of distal portions of diabetic flaps 7 days after surgery. Quantitative analysis of (B) surviving flap area, (C) capillary density, and (D) number of CD31 -
positive cells. Results are shown as means ± SEM. *P<0.01 vs. the PBS-treated and control groups (C and D), while *P<0.05 vs. the PBS-treated and
control groups (B). Scale bar =100 jam. HP = high-power field. DFO-treated (n = 20), PBS-treated (n = 16) and Blank group (n = 16).
doi:1 0.1 371 /journal. pone.01 0081 8.g004

increased by the administration of DFO. Furthermore, local
injection of DFO resulted in an increased mobilization of EPGs in
diabetic mice.

Necrosis of skin flaps in diabetic conditions has been attributed
to chronic hyperglycemia, which leads to defective angiogenesis in
tissues responding to low oxygen tension [9]. Thus, restoring
adequate response to hypoxia in ischemic tissue is of vital

importance to stimulate neovascularization in the flap. In the
present study, we showed that local administration of DFO
significantly increased the viability of random skin flaps in diabetic
mice. Meanwhile, within the DFO-treated diabetic flaps, an
elevated blood perfusion was detected, which was concordant with
enhanced capillary density determined by CD 31 immunofluores-
cence. Furthermore, mobilization of EPCs in the peripheral

Intact
Skin

Flap

P -actin

VEGF

PBS BLANK NDM DFO

HIF-1 a

PBS BLANK NDM DFO

Figure 5. Expression of VEGF and HIF-1 a in diabetic flaps evaluated by western blot 3 days after surgery. Expression of VEGF (A) and
HIF-1 oc (C) was significantly increased in the diabetic flaps of the DFO-treated group. Semi-quantitative analysis of western blots (B and D). *P<0.01 vs.
the PBS and untreated groups. NDM: Non-diabetic. n = 7.
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DFO PBS DFO PBS

Figure 6. Western-blot detection of VEGF and HIF-1a production in dermal fibroblasts cultured in normoxia (21% 0 2 ) or hypoxia
(1% 0 2 ) under different glucose concentrations in response to DFO administration for 72 hours. Addition of DFO in the culture medium
significantly improved the expression of (A) VEGF and (B) HIF-1 oc in dermal fibroblasts under hypoxia and high glucose conditions. Semi-quantitative
analysis of western blots (C and D). Results are shown as means ± SEM. *P<0.05. LG: Low Glucose; HG: High Glucose. n = 6.
doi:1 0.1 371 /journal. pone.01 0081 8.g006

circulation was greatly increased in DFO-treated diabetic mice
compared with the control groups. Taken together, these results
demonstrated that improved viability of diabetic skin flaps by local
administration of DFO was a consequence of stimulated
neovascularization.

As the key transcription factor that mediates the adaptive
response to hypoxia, HIF- 1 ex has been proved to be a key regulator
of angiogenic gene expression by binding to a conserved and
defined hypoxia response element in the genes activated by it

W Normal DM DFO+DM

Figure 7. Proportion of circulating EPCs in DFO-treated and
untreated diabetic mice, as determined by FACS analysis (A).

Flk1 + /CD11b~ cells represent circulating EPCs. (B) Quantitative analysis
of EPC mobilization are shown as means ± SEM. *P<0.05. DM: diabetic.
Normal indicates mice without any treatment. n = 5.
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[24,25]. However, it was found that high-glucose conditions
induced the production of methyglyoxal, leading to the modifica-
tion of HIF-1 ex coactivator p300, which attenuates the association
of p300 with HIF- lex, and therefore prevents HIF- 1 -mediated
gene transactivation. Therefore, DFO could attenuate methylgly-
oxal production, decrease the modification of p300 by methylgly-
oxal, and thus normalize hypoxic response of cells exposed to high
glucose environment by the correction of impaired HIF-lex/p300
association [3]. However, whether the improved neovasculariza-
tion induced by DFO in diabetic flaps is the result of increased
HIF- la accumulation remains unclear. In the present study, we
found that the expression of HIF- lex was greatly reduced in
diabetic flaps. With the local administration of DFO, the
expression of HIF- lex and VEGF recovered to a similar level as
that in non-diabetic mice, which was in accordance with improved
viability of skin flaps. It has been reported that hyperglycemia
attenuates VEGF production [2,3], and decreased VEGF levels
have been observed in the wounds of diabetic mice [2,26]. Thus,
with improved HIF- lex expression, VEGF production was restored
by local DFO injection.

The repair and regeneration of the vascular system requires
local vessel remodeling through vasculogenesis and angiogenesis
[15], during which EPGs are mobilized to the ischemic sites from
the bone marrow and take part in the formation of new blood
vessels in injured tissue [27]. Vasculogenesis requires a subtle
cascade of signaling events capable of mobilizing, homing and
retaining bone marrow-derived EPCs to induce neovascularization
in response to up-regulated HIF- lex and local VEGF production
[28]. However, in agreement with the present study, previous
studies have demonstrated that mobilization of EPCs is greatly
decreased in diabetic mice [29,30]. Therefore, the necrosis of
diabetic flaps may be secondary to decreased EPC recruitment as
a result of decreased HIF- lex and VEGF expression. The ability to
stabilize HIF- 1 ex with DFO administration and to retain a normal

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Effects of DFO on Diabetic Random Flap

hypoxia response is critical for the guidance and retention of
reparative EPCs. With DFO administration, angiogenesis in
ischemic diabetic random flaps was improved as a result of
stabilization of intracellular HIF-lot in response to hypoxia, by
which VEGF production and EPCs migration was enhanced [31].
These results were consistent with the findings by Chang et al,
who reported that DFO only promoted neovascularization in
ischemic areas, and did not produce any neovascularization in
uninjured skin [15].

Dermal fibroblasts play a critical role for regulating contraction,
extracellular matrix deposition and neovascularization in cutane-
ous wound repair. Lerman et al. revealed that dermal fibroblasts
from diabetic mice exhibited impaired migration, VEGF expres-
sion and response to hypoxia under hyperglycemia [32]. In the
present study, we observed that diabetic fibroblasts failed to
produce HIF-1(X and VEGF in response to hypoxia. However,
with the addition of DFO in the culture medium, expression of
HIF-1(X and VEGF was restored to the same levels as non-diabetic
fibroblasts. Thus, this in vitro study suggested that DFO performed
its angiogenic role through the increase of HIF-1(X and VEGF
expression in dermal fibroblasts in necrotic flaps in diabetic mice.

Many findings have reported that DFO could stabilize HIF- 1 a
and increase VEGF expression [11,15,33]. As a result, DFO
promoted neovascularization in normal skin flaps in aged mice
and enhanced wound healing in diabetic tissues [3,1 1,15]. To our
knowledge, this is the first report about the improvement of the

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survival of diabetic skin flaps by local administration of DFO. The
local delivery of DFO for the treatment of human diabetic necrotic
flaps may have significant clinical implications for diabetic patients
[34,35]. Of course, further studies should focus on DFO-mediated
cellular cascade involving eNOS, SDF-la, and PHD, which play
roles in the neovascularization in ischemic environment and in
diabetic-related complications [30,36,37].

Conclusion

In conclusion, our studies demonstrated that a local injection of
DFO could improve the viability of ischemic random skin flaps by
enhancing neovascularization in STZ-induced diabetic mice. With
DFO administration, HIF- 1 a stability was increased and led to an
increased expression of VEGF, as well as promoted the
mobilization of EPC in the peripheral circulation. In addition,
DFO restored HIF- 1 cx-mediated VEGF expression in diabetic
fibroblasts in response to hypoxia. Thus, local administration of
DFO may be a safe, easy and alternative treatment for necrotic
random skin flap in diabetic patients.

Author Contributions

Conceived and designed the experiments: GW LG. Performed the
experiments: YG YZ. Analyzed the data: GW YZ ZX. Contributed
reagents/materials/analysis tools: GL. Wrote the paper: GW YC LG.

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